Catalytic antioxidants and methods of use

ABSTRACT

The invention provides small molecules that act as catalytic antioxidants and methods of use thereof. The compounds can repeatedly bind and destroy reactive oxygen species by serving as substates for enzymes of the methionine sulfoxide reductase (Msr) class. Some embodiments of the catalytic antioxidant compounds are derived from drugs with anti-inflammatory activity due to inhibition of cyclooxygenase enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority of U.S. provisionalpatent application No. 60/429,269 filed on Nov. 26, 2002.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] Not applicable.

FIELD OF THE INVENTION

[0003] The invention relates to the fields of biochemistry,pharmacology, and medicine.

[0004] More particularly, the invention relates to methods andcompositions for promoting health and increasing longevity by reducingoxidative damage to cells and tissues.

BACKGROUND

[0005] Oxygen is involved in a wide range of normal metabolic reactionsand is essential for the survival of all aerobic organisms, includinghuman beings. Reactive oxygen species (ROS), such as superoxide, areproduced in abundance as a byproduct of the incomplete reduction ofoxygen that has entered the respiratory chain. Superoxide is theprecursor of other damaging oxygen species including hydrogen peroxide,the hypochlorite ion and the hydroxyl radical. Oxidase enzymes in cellssuch as phagocytes and nitric oxide synthases are other sources of ROS.

[0006] While low levels of ROS are present under normal physiologicalconditions, in excess, ROS can cause oxidative damage to cells andtissues by, for example, oxidizing cellular macromolecules such asnucleic acids, lipids and proteins. Cumulative damage to cells in thismanner can result in pathology. Not surprisingly then, oxidative damagehas been implicated in a wide variety of diseases and conditionsincluding chronic obstructive lung disorders such as smoker's emphysema,reperfusion damage, neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS),heart attacks, stroke, several autoimmune diseases, and aging.

[0007] Regarding the latter, oxidative damage to cellular macromoleculeshas been postulated to accelerate the aging process and shortenlifespan. For example, the level of oxidized methionine in proteins inan animal has been observed to increase with the age of the animal.Moreover, in Drosophila, greater resistance to ROS via over-expressionof superoxide dismutase and catalase has been correlated with longerlifespan, whereas genetic disruption of superoxide dismutase andcatalase has been correlated with shorter lifespan.

[0008] Although cells have evolved their own enzymatic antioxidantsystems (e.g., superoxide dismutase, catalase, and peroxidase) toneutralize ROS, such systems may not function at ideal levels tominimize the rate of aging and the development of disease. Accordingly,there is a clear need for non-naturally occurring compositions andmethods that reduce oxidative damage to cells. One approach to increasethe antioxidant activity in cells is to provide cells with compoundsthat directly scavenge ROS, e.g., vitamins C, E, and A, glutathione,ubiquinone, uric acid, carotenoids, and the like. Such conventionalantioxidant compounds, however, lose activity after neutralizing onlyone or two ROS molecules. They are thus limited by the relatively smallquantities of ROS that they can destroy.

SUMMARY

[0009] The invention relates to the development of methyl sulfoxide ormethyl sulfide containing catalytic antioxidants that can repeatedly beoxidized by a ROS, reduced back to an unoxidized form, and oxidizedagain by a ROS. Unlike a conventional antioxidant molecule, a singlecatalytic antioxidant molecule of the invention can neutralize amultitude of different ROS molecules.

[0010] The regenerative capacity of the catalytic antioxidant moleculesof the invention is based on their ability to act as substrates for themethionine sulfoxide reductase (Msr) class of enzymes. Among the variousamino acids found in proteins, methionine (Met) is one of the mostsusceptible to oxidation. Oxidation of methionine by ROS yieldsmethionine sulfoxide [Met(O)]. The Msr enzymes, including MsrA and MsrBprominent in virtually all cells, including mammalian cells, act asrepair enzymes that catalyze the reversal of the oxidation reaction,reducing Met(O) back to methionine. In addition to reducing methionine,MsrA and several other forms of Msr enzymes known in bacteria can reducea variety of other substrates, but in all cases the core functionalgroup recognized by the enzymes is a methyl sulfoxide moiety. Byreducing methyl sulfoxide moieties back to methyl sulfide, the Msrenzymes repair damaging oxidation reactions to methionine in proteins.In addition the methionine residues in proteins, via cyclic oxidationand reduction by the Msr system, can act as scavengers of ROS. In theseways the Msr system is believed to contribute to the longevity andhealth of cells by conferring resistance to ROS (reviewed in Weissbachet al., Archiv. Biochem. Biophys. 397:172-178, 2002).

[0011] The catalytic antioxidants of the invention are small moleculesthat act as substrates for Msr enzymes. A scheme of interaction of thecompounds of the invention with the Msr pathway is showndiagrammatically in FIG. 1. Methyl sulfide groups on the antioxidantcompounds can react with reactive oxygen species (ROS) such assuperoxide or hydrogen peroxide to form methyl sulfoxides, for examplemethionine sulfoxide, which occurs in proteins and in the free form incells. Upon trapping and destruction of the ROS by a catalyticantioxidant compound of the invention, the methyl sulfoxide formedthereon can serve as a substrate for one or more Msr enzymes.Nucleophilic attack of the methyl sulfoxide by a cysteine residue in theMsr enzyme leads to transfer of the oxygen from the compound to theenzyme, reducing the compound back to its unoxidized state (FIG. 1). Thecompounds, thus regenerated, are available for repeated reuse asantioxidants. Thus, the catalytic antioxidant compounds of the inventionfunction not only as typical ROS scavengers, but also regeneratethemselves by harnessing the catalytic action of the Msr enzymes.

[0012] Accordingly, in one aspect, the invention features non-naturallyoccurring (or purified, naturally occurring) compounds including atleast one methyl sulfide or methyl sulfoxide moiety, the compounds beinga substrate for at least one MsrA enzyme and at least one MsrB enzyme,or a pharmaceutically acceptable salt thereof. Certain embodiments ofthe compounds are based on a backbone derived from the chemicalstructure of sulindac(1(Z)-5-fluoro-2-methyl-1[[4-(methylsulfinyl)phenyl)methylene]-1H-indenyl-3-aceticacid).

[0013] Other embodiments of the compounds are non-naturally occurring(or purified, naturally occurring) compounds including at least onemethyl sulfide or methyl sulfoxide moiety, the compounds being asubstrate for at least one Msr enzyme and having a backbone not based onsulindac. Various embodiments of these compounds have a backbone basedon several known cyclooxygenase (COX) inhibitors, including acetylsalicylic acid, mefenamic acid, ibuprofen, indomethacin, and rofecoxib(Vioxx®). The invention also includes compositions based on thesecompounds in a pharmaceutically acceptable carrier.

[0014] In another aspect, the invention provides a method for reducing,preventing or reversing oxidative damage in a cell. The method includesthe steps of: (a) providing a non-naturally occurring (or purified,naturally occurring) compound including in its chemical structure atleast one methyl sulfide or methyl sulfoxide moiety, the compound beinga substrate for at least one Msr enzyme; (b) providing a cell expressingat least one Msr enzyme, the cell containing or being exposed toreactive oxygen species; and (c) contacting the cell with an amount ofthe compound sufficient to reduce, prevent, or reverse oxidative damagein the cell by the reactive oxygen species.

[0015] The cell can be within an animal subject, such as a human being.The animal subject can have a condition or disorder associated withoxidative damage. The disorder can involve degeneration of a nerve cell.The condition affecting the subject can be age-related.

[0016] Yet another embodiment of the invention is a method for extendingthe lifespan of an animal. The method involves administering to theanimal a therapeutically effective amount of a non-naturally occurring(or purified, naturally occurring) compound including at least onemethyl sulfide or methyl sulfoxide moiety, the compound being asubstrate for at least one Msr enzyme.

[0017] As used herein, the terms “methionine moiety” and “methionineanalog” include all structures encompassed by general methionine formula1 described herein, including selenomethionine derivatives.

[0018] As used herein, the term “catalytic antioxidant” refers to anon-naturally occurring (or purified, naturally occurring) antioxidantcompound that can be enzymatically regenerated after it is oxidized byan oxidizing agent (for example a ROS) such that each equivalent ofantioxidant compound can destroy more than one equivalent of theoxidizing agent.

[0019] Unless otherwise defined, all technical terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. The particular embodiments discussedbelow are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions willcontrol.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a schematic diagram showing the mechanism of action of acatalytic anti-oxidant, according to an embodiment of the invention.

[0021]FIG. 2 is a schematic diagram showing the cycle of catalyticantioxidant activity of sulindac, catalyzed by an MsrA enzyme, accordingto an embodiment of the invention.

[0022]FIGS. 3A and 3B is two graphs showing kinetics of sulindac sulfideproduction by MsrA, according to an embodiment of the invention.

[0023]FIGS. 4A and 4B is a schematic diagram showing chemical syntheticpathways for making methionine derivatives of sulindac (compounds 2a and3a), according to an embodiment of the invention.

[0024]FIGS. 5A and 5B is a schematic diagram showing chemical syntheticpathways for making methionine derivatives of sulindac (compounds 4a and5a), according to an embodiment of the invention.

[0025]FIGS. 6A and 6B is a schematic diagram showing chemical syntheticpathways for making catalytic antioxidants based on salicylic acid andmefenamic acid (compounds 6a and 7a, respectively), according to anembodiment of the invention.

[0026] FIGS. 7A-C is a schematic diagram showing chemical syntheticpathways for making catalytic antioxidants based on ibuprofen,indomethacin and Vioxx® (compounds 8a, 9a, and 10a, respectively),according to an embodiment of the invention.

[0027]FIG. 8 shows a NMR spectrum of compound 2a of the invention.

[0028]FIG. 9 is a micrograph of a TLC plate showing the presence ofreduction products of sulindac (S) and sulindac methionine sulfoxide(SMO) following incubation with MsrA and MsrB enzymes. Resultsdemonstrate that S is a substrate for MsrA and that SMO is a substratefor both MsrA and MsrB.

[0029]FIG. 10 is a graph showing enhanced survival of sulindac-treatedflies exposed to oxidative stress induced by paraquat.

[0030]FIG. 11 is a graph showing enhanced survival of G93A transgenicmice over expressing a mutant superoxide dismutase withneurodegenerative disease treated with sulindac.

[0031]FIG. 12 is a graph showing enhanced motor performance ofsulindac-treated transgenic G93A mice.

[0032]FIG. 13 is a graph showing neuronal cell counts in sections ofspinal cords of G93A mice. Neuronal cell survival is significantlyhigher in animals receiving sulindac.

DETAILED DESCRIPTION

[0033] The invention encompasses compositions and methods relating tocatalytic antioxidants useful in reducing or preventing oxidative damagein cells. The antioxidant compounds contain active sites that captureROS. The antioxidant ability of the compounds is regenerated followingcapture of ROS by interaction with enzymes of the Msr class that reducemethyl sulfoxide moieties back to the methyl sulfide.

[0034] The below described preferred embodiments illustrate variouscompositions and methods within the invention. Nonetheless, from thedescription of these embodiments, other aspects of the invention can bemade and/or practiced based on the description provided below.

Biological Methods

[0035] Methods involving conventional chemistry, cell biology andmolecular biology techniques are described herein. Such techniques aregenerally known in the art and are described in detail in methodologytreatises such as Classics in Total Synthesis. Targets, Strategies,Methods, K. C. Nicolaou and E. J. Sorensen, VCH, New York, 1996; and TheLogic of Chemical Synthesis, E. J. Coney and Xue-Min Cheng, Wiley &Sons, New York, 1989. Molecular biological and cell biological methodsare described in treatises such as Molecular Cloning: A LaboratoryManual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocolsin Molecular Biology, ed. Ausubel et al., Greene Publishing andWiley-Interscience, New York, 1992 (with periodic updates).

Catalytic Antioxidants having Methyl Sulfide or Methyl Sulfoxide Groups

[0036] The invention provides small molecules containing at least one(e.g., 1, 2, 3 or more) methyl sulfoxide or methyl sulfide group thatcan enter cells and prevent oxidative damage by a catalytic antioxidantmechanism. The methyl sulfide group on the compounds reacts with ROS,forming methyl sulfoxide. The methyl sulfoxide-bearing compounds, inturn, act as substrates for Msr enzymes which reduce the compounds andthereby regenerate their antioxidant properties. These compounds can beadministered to cells or animals to reduce cellular damage caused byROS.

[0037] Referring to FIG. 1, these compounds serve 1) as ROS scavengers(antioxidants) by virtue of the active groups within their structuresthat destroy or react with ROS, and 2) as catalytic antioxidants byacting as substrates for Msr enzymes that reduce the oxidized compoundsback to the unoxidized form capable of further reaction with ROS. Thecatalytic nature of the antioxidant compounds of the invention is due totheir ability to serve as substrates for Msr enzymes. The corefunctional group recognized by these enzymes is methyl sulfoxide. In thecase of N-methionine-containing peptide and protein substrates, thisfunctional group is contained within the amino acid methionine.

[0038] Any compound having a methyl sulfide or methyl sulfoxidefunctional group that is a substrate for a Msr enzyme can be used.Sulindac, a non-steroidal anti-inflammatory drug and COX inhibitor, isone example of a methyl sulfoxide-containing compound that serves as asubstrate for Msr enzymes. Sulindac is a pro drug, and is only active asa COX inhibitor when the methyl sulfoxide moiety on the molecule isreduced to the sulfide. Heretofore, sulindac was not known to act as asubstrate for a Msr. FIG. 2 shows the reduction of sulindac to sulindacsulfide, catalyzed by Msr. As described below, sulindac was tested as asubstrate against six known members of the Msr family identified inbacteria (E. coli) and against Msr enzymes present in mammalian (bovine)tissues. MsrA and a membrane-associated Msr of bacteria were shown to beable to reduce sulindac to the active sulfide. In mammalian tissues,reduction of sulindac was primarily attributable to the activity ofMsrA.

[0039] As further described below, sulindac administration (1) protectedDrosophila against the damage from paraquat-induced ROS production, (2)prolonged the survival of spinal cord motor neurons in mice with aneurodegenerative disease caused by oxidative damage, and (3) extendedthe lifespan of the foregoing mice.

Methionine-Based Catalytic Antioxidants

[0040] In one aspect, the invention provides catalytic antioxidantcompounds having methionine moieties or analogs of methionine. Suchcompounds are substrates for Msr enzymes that recognize the methylsulfoxide functional group in methionine (for example, MsrA and MsrB).The methionine moiety or analog found in the methionine-containingembodiments of the compounds has the following general structure:

[0041] Groups R₁, R₂, R₃, and X in general structure 1 are defined asfollows:

[0042] R₁ may be CH (of either R or S configuration).

[0043] R₂ may be a normal or branched alkyl or fluoroalkyl group having1 to 6 carbons.

[0044] R₃ may be ethyl or preferably methyl, or a fluorinated derivativethereof.

[0045] X may be either S or Se in any oxidation state.

[0046] As used herein, the terms “methionine moiety” and “methionineanalog” include all structures encompassed by general formula 1,including selenomethionine analogs of methionine. General structure 1also includes esters and salts of the carboxylic acid. Oligopeptidescontaining methionine for attachment to small molecules are alsoencompassed by the invention.

Methionine-Based Catalytic Antioxidants Derived from COX Inhibitors

[0047] Inflammation and oxidative damage are known to coexist in manydisease states and degenerative conditions. Accordingly, particularlypreferred embodiments of the methionine-containing compounds of theinvention are derivatives of anti-inflammatory agents such as COXinhibitors. Specific examples of such compounds, employing scaffoldsbased on several COX inhibitors, and methods for their synthesis areprovided in the examples below. Exemplary compounds include thosederived from the following scaffolds:

[0048] sulindac; acetyl salicylic acid (ortho-acetoxybenzoic acid),mefenamic acid (2-[(2,3-Dimethylphenyl)amino]benzoic acid); ibuprofen(α-methyl-4-(2-methylpropyl)-benzeneacetic acid); indomethacin(1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole-3-acetic acid); androfecoxib (4-[4-(methylsulfonyl)phenyl]-3-phenyl- 2(5H)-furanone, forexample, Vioxx®, sold by Merck) and celecoxib(4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-benzenesulfonamide,for example, Celebrex® sold by Pfizer.

[0049] Embodiments of the invention that are sulindac derivatives canhave the following general formulas 2-5:

[0050] Groups R₁, R₂, R₃, R₄, R₅, R₆, and X in general formulas 2, 3, 4and 5 are defined as follows:

[0051] R₁ may be CH (of either R or S configuration).

[0052] R₂ may be a normal or branched alkyl or fluoroalkyl group having1 to 6 carbons.

[0053] R₃ may be ethyl or preferably methyl, or a fluorinated derivativethereof.

[0054] R₄ may be a hydrogen or a normal or branched alkyl group having 1to 6 carbons.

[0055] R₅ may be a CH (of either R or S configuration).

[0056] R₆ is a may be a hydrogen or a normal or branched alkyl orfluoroalkyl group having 1 to 6 carbons.

[0057] R₇ may be a nitrogen (with substituent R₄ as defined above), a CH(of either R or S configuration), or a normal or branched alkyl orfluoroalkyl group having 1 to 6 carbons.

[0058] X may be either S or Se in any oxidation state.

[0059] General structures 2, 3, 4 and 5 also include esters and salts ofthe carboxylic acid group. The invention also encompasses sulindacderivatives containing oligomeric methionine moieties and analogs.[

[0060] Embodiments of the invention that are acetyl salicylic acidderivatives can have the following general formula:

[0061] The aromatic ring of general structure 6 may contain one or morenitrogen atoms (for example pyridine or pyrazine). The aromatic carboxylgroup in general structure 6 may be oriented ortho, meta, orpara to themethionine-based moiety. Groups R₁, R₂, R₃, R₄, R₅, and

[0062] X in the general structure are defined as follows:

[0063] R₁ may be CH (of either R or S configuration).

[0064] R₂ may be a normal or branched alkyl or fluoroalkyl group having1 to 6 carbons.

[0065] R₃ may be ethyl or preferably methyl or a fluorinated derivativethereof.

[0066] R₄ may be a hydrogen or a normal or branched alkyl group having 1to 6 carbons.

[0067] R₅ may be a nitrogen (with substituent R₄ as defined above), anoxygen, or a sulfur.

[0068] X may be either S or Se in any oxidation state.

[0069] General structure 6 also includes esters and salts of thecarboxylic acid group. The invention also encompasses acetyl salicylicacid derivatives containing oligomeric methionine moieties and analogs.

[0070] Embodiments of the invention that are mefenamic acid derivativescan have the following general formula:

[0071] Both aromatic rings of general structure 7 may contain one ormore nitrogen atoms (for example pyridine or pyrazine). The aromaticcarboxyl group in general structure 7 may be oriented ortho, meta,orpara to the aniline nitrogen. Groups R₁, R₂, R₃, R₄, and X in thegeneral structure are defined as follows:

[0072] R₁ may be CH (of either R or S configuration).

[0073] R₂ may be a normal or branched alkyl or fluoroalkyl group having1 to 6 carbons.

[0074] R₃ may be ethyl or preferably methyl or a fluorinated derivativethereof.

[0075] R₄ may be a hydrogen or a normal or branched alkyl group having 1to 6 carbons.

[0076] X may be either S or Se in any oxidation state.

[0077] General structure 7 also includes esters and salts of thecarboxylic acid group. The invention also encompasses mefenamic acidderivatives containing oligomeric methionine moieties and analogs.

[0078] Embodiments of the invention that are ibuprofen derivatives canhave the following general formula:

[0079] The aromatic ring of general structure 8 may contain one or morenitrogen atoms (for example pyridine or pyrazine). The sec-butyl groupin general structure 8 may be oriented ortho, meta, orpara to themethionine-based moiety. Groups R₁, R₂, R₃, R₄, R₅, and X in the generalstructure are defined as follows:

[0080] R₁ may be CH (of either R or S configuration).

[0081] R₂ may be a normal or branched alkyl or fluoroalkyl group having1 to 6 carbons.

[0082] R₃ may be ethyl or preferably methyl or a fluorinated derivativethereof.

[0083] R₄ may be a hydrogen or a normal or branched alkyl group having 1to 6 carbons.

[0084] R₅ may be a CH (of either R or S configuration).

[0085] X may be either S or Se in any oxidation state.

[0086] General structure 8 also includes esters and salts of thecarboxylic acid group. The invention also encompasses ibuprofenderivatives containing oligomeric methionine moieties and analogs.

[0087] Embodiments of the invention that are indomethacin derivativescan have the following general formula:

[0088] Groups R₁, R₂, R₃, R₄, R₅, R₆, R₇ and X in general structure 9are defined as follows:

[0089] R₁ may be CH (of either R or S configuration).

[0090] R₂ may be a normal or branched alkyl or fluoroalkyl group having1 to 6 carbons.

[0091] R₃ may be ethyl or preferably methyl or a fluorinated derivativethereof.

[0092] R₄ may be a hydrogen or a normal or branched alkyl group having 1to 6 carbons.

[0093] R₅ may be a CH (of either R or S configuration).

[0094] R₆ is a may be a hydrogen or a normal or branched alkyl orfluoroalkyl group consisting of 1 to 6 carbons.

[0095] R₇ may be any halogen oriented ortho, meta, orpara to thecarbonyl group.

[0096] X may be either S or Se in any oxidation state.

[0097] General structure 9 also includes esters and salts of thecarboxylic acid group. The invention also encompasses indomethacinderivatives containing oligomeric methionine moieties and analogs.

[0098] Embodiments of the invention that are Vioxx® derivatives can havethe following general formula:

[0099] The lactone ring in general structure 10 may be oriented ortho,meta, orpara to sulfonyl group. Groups R₁, R₂, R₃, R₄, and X in generalstructure 10 are defined as follows:

[0100] R₁ may be CH (of either R or S configuration).

[0101] R₂ may be a normal or branched alkyl or fluoroalkyl group having1 to 6 carbons.

[0102] R₃ may be ethyl or preferably methyl or a fluorinated derivativethereof.

[0103] R₄ may be a hydrogen or a normal or branched alkyl group having 1to 6 carbons.

[0104] X may be either S or Se in any oxidation state.

[0105] Ar may be phenyl, alkyl and halogen substituted phenyl, andheteroaromatic compounds.

[0106] General structure 10 also includes esters and salts of thecarboxylic acid group. The invention also encompasses Vioxx® derivativescontaining oligomeric methionine moieties and analogs.

Testing of Catalytic Antioxidant Compounds

[0107] The ability of any given molecule having a chemical structureincluding at least one methyl sulfoxide- and/or methylsulfide-containing moiety, or at least one methionine and/or methioninesulfoxide moiety to act as a catalytic antioxidant can be determinedempirically.

[0108] For example, a molecule containing a methyl sulfoxide group to betested (i.e., a test molecule) can be subjected to an enzymatic assaythat indicates if the test molecule can serve as a substrate for MsrA,MsrB or other members of the Msr family (see, for instance, the NADPHassay described in Example 1, and the extraction assay described inExample 2, below). A test molecule can also be subjected to an assaythat indicates the molecule's ability to increase resistance tooxidative stress in cells in vitro (for example PC-12 cells subjected toinsult with MPP+) or in an animal subject, for example, Drosophila or amammalian model of oxidative damage. See, for instance, the assaysdescribed in Examples 7, 8 and 9 below.

Preventing/Reversing Oxidative Damage In A Cell

[0109] The catalytic antioxidant compounds of the invention can be usedto reduce, prevent or reverse oxidative damage in a cell (for example, acell in an animal). In this method, a non-naturally occurring catalyticantioxidant compound is brought into contact with the cell. Afterentering the interior of the cell, the compound, if in the reduced(sulfide) form, will be oxidized to the sulfoxide by ROS (i.e., act as aROS scavenger). Subsequent reduction catalyzed by an Msr enzyme willregenerate the original sulfide. If the test molecule contains a methylsulfoxide moiety, it will be reduced to the sulfide by the Msr systemwithin the cell and subsequently act as an antioxidant. With either thesulfide or the sulfoxide as the test molecule, the oxidation/reductioncycle will permit the compound to destroy ROS catalytically, as shown inFIG. 1.

[0110] The effectiveness of particular compounds can be assessed usingconventional in vitro and in vivo assays, for example, determining acell's, or an animal's response to a challenge with an agent thatproduces ROS. For instance, to assess a test molecule for the ability toprevent oxidative damage caused by ROS in a cell, cells can be culturedby conventional means and challenged with an agent that produces ROSwithin the cells. An exemplary cellular system for testing the effect ofROS damage in nerve cells, for example, is an assay employing PC-12cells subjected to insult with MPP+, an agent that generates superoxideand other oxygen radicals. To assess the efficacy of a test compound inan animal, Drosophila melanogaster (fruit fly) is an excellent animalmodel. The flies can be treated with an agent that produces ROS (forexample, paraquat) and then fed with a diet containing the test moleculeand monitored for their survival, compared to control flies receivingParaquat alone. Mammalian models of oxidative damage are also well knownand include inter alia a transgenic mouse model of amyotrophic lateralsclerosis (ALS) based on a mutation in the superoxide dismutase (SODI)gene.

Animal Subjects

[0111] Because oxidative damage to cells is a ubiquitous phenomenon, theinvention is believed to be compatible with any animal subject. Anon-exhaustive list of examples of such animals includes mammals such asmice, rats, rabbits, goats, sheep, pigs, horses, cattle, dogs, cats, andprimates such as monkeys, apes, and human beings. Those animal subjectsthat have a disease or condition that relates to oxidative damage arepreferred for use in the invention as these animals may have thesymptoms of their disease reduced or even reversed. In particular, humanpatients suffering from inflammation, chronic obstructive lung diseasessuch as emphysema, reperfusion damage after heart attack or stroke,neurodegenerative diseases (for example, Parkinson's disease,Alzheimer's disease, and ALS), autoimmune diseases such as rheumatoidarthritis, lupus, and Crohn's disease, conditions related to prematurebirth, conditions caused by exposure to ultraviolet light, andage-related conditions (as but one example, age-related degenerativeconditions of the eye including age-related macular degeneration andcataract formation) are suitable animal subjects for use in theinvention. In the experiments described herein, animals used fordemonstration of beneficial effects of protection against ROS damage bythe compounds of the invention are the fruit fly and the mouse.Nonetheless, by adapting the methods taught herein to other methodsknown in medicine or veterinary science (for example, adjusting doses ofadministered substances according to the weight of the subject animal),the compounds and compositions of the invention can be readily optimizedfor use in other animals.

Administration of Compositions

[0112] The catalytic antioxidant compositions of the invention may beadministered to animals including humans in any suitable formulation.For example, the compositions may be formulated in pharmaceuticallyacceptable carriers or diluents such as physiological saline or abuffered salt solution. Suitable carriers and diluents can be selectedon the basis of mode and route of administration and standardpharmaceutical practice. A description of other exemplarypharmaceutically acceptable carriers and diluents, as well aspharmaceutical formulations, can be found in Remington's PharmaceuticalSciences, a standard text in this field, and in USP/NF. Other substancesmay be added to the compositions to stabilize and/or preserve thecompositions, or enhance the activity of the Msr system. One suchenhancing substance could be nicotinamide which is part of the molecule,NADPH, that supplies the reducing power to the reaction catalyzed by themembers of the Msr family.

[0113] The compositions of the invention may be administered to animalsby any conventional technique. Such administration may be oral orparenteral (for example, by intravenous, subcutaneous, intramuscular, orintraperitoneal introduction). The compositions may also be administereddirectly to the target site by, for example, surgical delivery to aninternal or external target site, or by catheter to a site accessible bya blood vessel. Other methods of delivery, for example, liposomaldelivery or diffusion from a device impregnated with the composition,are known in the art. The compositions may be administered in a singlebolus, multiple injections, or by continuous infusion (for example,intravenously or by peritoneal dialysis). For parenteral administration,the compositions are preferably formulated in a sterilized pyrogen-freeform.

[0114] Compositions of the invention can also be administered in vitroto a cell (for example, to prevent oxidative damage during ex vivo cellmanipulation, for example of organs used for organ transplantation or inin vitro assays) by simply adding the composition to the fluid in whichthe cell is contained.

Effective Doses

[0115] An effective amount is an amount which is capable of producing adesirable result in a treated animal or cell (for example, reducedoxidative damage to cells in the animal or cell). As is well known inthe medical and veterinary arts, dosage for any one animal depends onmany factors, including the particular animal's size, body surface area,age, the particular composition to be administered, time and route ofadministration, general health, and other drugs being administeredconcurrently. It is expected that an appropriate dosage for parenteralor oral administration of compositions of the invention would be in therange of about 1 μg to 100 mg/kg of body weight in humans. An effectiveamount for use with a cell in culture will also vary, but can be readilydetermined empirically (for example, by adding varying concentrations tothe cell and selecting the concentration that best produces the desiredresult). It is expected that an appropriate concentration would be inthe range of about 0.0001-100 mM. More specific dosages can bedetermined by the method described below.

[0116] Toxicity and efficacy of the compositions of the invention can bedetermined by standard pharmaceutical procedures, using cells in cultureand/or experimental animals to determine the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose that effects the desiredresult in 50% of the population). Compositions that exhibit a largeLD₅₀/ED₅₀ ratio are preferred. Although less toxic compositions aregenerally preferred, more toxic compositions may sometimes be used in invivo applications if appropriate steps are taken to minimize the toxicside effects.

[0117] Data obtained from cell culture and animal studies can be used inestimating an appropriate dose range for use in humans. A preferreddosage range is one that results in circulating concentrations of thecomposition that cause little or no toxicity. The dosage may vary withinthis range depending on the form of the composition employed and themethod of administration.

EXAMPLES

[0118] The present invention is further illustrated by the followingspecific examples, which should not be construed as limiting the scopeor content of the invention in any way.

Example 1 Sulindac is a Substrate For MsrA Enzyme

[0119] The enzyme methionine sulfoxide reductase (MsrA) is known toexhibit specificity for substrates that contain a methyl sulfoxidegroup. This example provides evidence that sulindac, a known antioxidantcontaining a methyl sulfoxide moiety, can act as a substrate for MsrA.

[0120] Materials and Methods.

[0121] Reductase assay. With a purified Msr enzyme, sulindac reductioncan be measured by a modified NADPH oxidation assay. Reaction mixtureswere prepared containing 50 mM Tris-Cl pH 7.4, 15 μg of E. colithioredoxin, 1 μg E. coli thioredoxin reductase, 100 nmoles of NADPH, 1μmole of sulindac and 100-400 ng of MsrA in a final volume of 500 μl.Incubations were performed at 37° C. for various times.

[0122] The amount of product (sulindac sulfide) synthesized wasdetemined by measuring the oxidation of NADPH spectrophotometrically at340 nm. Because sulindac absorbs very strongly at this wavelength, theloss of absorbance at 340 nm could not be measured directly. Toaccomplish this, the sulindac and sulindac sulfide were removed from theincubations by extraction with ethyl acetate as follows. At the end ofincubation, 500 μl of 0.5 M Bis-Tris-Cl pH 5.5 and 3 ml of ethyl acetatewere added. The tubes were mixed (vortexed) for 5 seconds (3 times).After separation, the organic phase was removed and another 3 ml ofethyl acetate were added. After mixing the organic phase was againremoved. The two extractions essentially removed all of the sulindac andsulindac sulfide, leaving the NADPH in the aqueous phase, which wasmeasured at 340 nm. The loss of absorption at 340 nm, dependent onsulindac, is a measure of sulindac reduction. (Δ 0.062 at 340 nm=10nmoles of sulindac sulfide formed).

[0123] Results.

[0124] The results of a reductase assay using MsrA from E. coli aresummarized below in Table 1. TABLE 1 Reduction of Sulindac to SulindacSulfide by MsrA Sulindac MsrA Sulindac Thioredoxin MetS(O) Time Δsulfide Tube # (100 ng/μl) (0.2M) (5 μg/μl) (0.2M) (min) OD₃₄₀ OD₃₄₀(nmol) 1 8 μl — 3 μl — 0.657 0 2 — 5 μl 3 μl — 0.660 0 3 1 μl 5 μl 3 μl— 20 0.622 0.038 5.8 4 2 μl 5 μl 3 μl — 20 0.586 0.074 11.2 5 4 μl 5 μl3 μl — 20 0.522 0.138 21.0 6 2 μl 5 μl — — 20 0.684 7 2 μl 5 μl 3 μl —10 0.626 0.034 5.2 8 2 μl 5 μl 3 μl — 30 0.531 0.129 19.4

[0125] The results show that sulindac was reduced in a time- andconcentration-dependent manner by MsrA enzyme.

Example 2 Sulindac is a Substrate for Msr Enzymes in Bacteria andMammals

[0126] This example demonstrates that sulindac is a substrate for MsrAand membrane-bound Msr in E. coli and for MsrA and possibly other Msrenzymes in mammalian tissues.

[0127] Material and Methods.

[0128] Chemicals, enzymes and substrates. Sulindac (S), sulindac sulfide(SS) and all other chemicals and E. coli thioredoxin reductase wereobtained from Sigma Chemicals (St. Louis, Mo.), unless noted otherwise.Thioredoxin (from E. coli) was purchased from Promega (Madison, Wis.).N-acetyl-³H-met-R, S—(O), met-R—(O), met-S—(O) DABS-met-R—(O) andDABS-met-S—(O) were prepared as previously described (Brot N. et al.,Anal. Biochem. 122 (1982) 291-294; Lavine, F. T. J. Biol. Chem. 169(1947) 477-491; Minetti G. et al., Ital. J. Biochem. 43 (1994) 273-283).

[0129] Bacterial enzymes. Recombinant MsrA and MsrB from Escherichiacoli were obtained as described previously (Grimaud, R.et al., J. Biol.Chem. 276 (2001) 48915-48920; Rahman, M. A. et al., Cellular & MolecularBiology 38 (1992) 529-542). Partially purified DEAE fractions offree-S-Msr (fSMsr), free-R Msr (fRMsr) and MsrA1, and a membrane vesicleassociated Msr (mem-R,S-Msr) were prepared from an E. coli MsrA/B doublemutant as described (Etienne, F. et al., Biochem. & Biophys. Res. Comm.300 (2003) 378-382; Spector, D. et al., Biochem. & Biophys. Res. Comm.302 (2003) 284-289). The enzyme preparations had specific activitiessimilar those reported earlier.

[0130] Mammalian enzymes. Calf liver, kidney and brain extracts wereprepared at 4° C. Thirty grams each of calf tissue (liver, kidney,brain) were minced using a hand-held homogenizer in 5 volumes of bufferA containing 250 mM sucrose, 10 mM Tris-Cl pH 7.4 and 1 mM EDTA. Thehomogenates were dounced (6 strokes) and spun at 1,500×g for 10 minutesand the pellet was discarded. The supernatants (S-10) were spun at10,000×g for 10 minutes. The S-10 supernatants were centrifuged at100,000×g for 12 hours and the resulting pellets and supernatants(S-100) were saved. The S100 pellets were suspended in cold buffer A andcentrifuged at 100,000×g for 4 hours. The washed microsomal pellets(containing all of the ribosomes) were suspended in 2 ml of buffer A(S-100 pellet).

[0131] To prepare mitochondria, the S10 pellets were suspended in 20 mlbuffer A. The suspension was layered on top of a discontinuous Ficollgradient made up of an equal volume of 12% Ficoll in buffer A (lowerlayer) and 7.5% Ficoll in buffer A (upper layer). The tubes werecentrifuged at 24,000×g for 24 min. The pellets were resuspended inbuffer A and centrifuged at 20,000×g for 15 min. The pellets (containingmitochondria) were suspended in 2 ml of buffer A. All fractions werestored at −80° C.

[0132] Reductase assay and quantitation of sulindac sulfide formed. Withcrude cellular fractions when there is a large amount of NADPHoxidation, the NADPH assay described in Example 1 above cannot be used.For use with crude cellular fractions, an extraction assay was developedbased on the ability of sulindac sulfide to be extracted into benzene.The reaction mixture for the reduction of sulindac to sulindac sulfidecontained in a total volume of 30 μl: 100 mM Tris-Cl, pH 7.4; 0.6 μmolesglucose-6-phosphate; 50 ng glucose-6-phosphate dehydrogenase; 30 nmolesNADPH; 2.5 μg thioredoxin, 1 μg thioredoxin reductase, 50 μmolessulindac and varying amount of Msr enzymes. Unless stated otherwise,incubations were for 1 hour at 37° C. At the end of the incubation 370μl of 25 mM Tris-Cl pH 8.0, 100 μl acetonitrile and 1 ml of benzene wereadded to each tube. After vortexing for 30 seconds and spinning for 1min at room temperature, the benzene phase was removed and the opticaldensity was read at 350 nm. Fifty nmoles of SS or S, when carriedthrough the extraction procedure, gave optical density readings of 0.910and 0.030, respectively. Under these conditions, virtually all of the SSwas extracted into the benzene, while about 2.5 % of S was extracted. Insome experiments using calf tissue extracts, the standard 30 μl reactionmixture volume was tripled (90 μl) to obtain statistically significantvalues. The extraction assay was not altered except for reduction of theTris buffer volume to 310 μl.

[0133] To remove the S epimer of sulindac, the sulindac (R, S mixture)was incubated with excess MsrA ( 4 μg) and DTT for 60 minutes, or untilthe reaction reached completion. Upon completion, any further reductionseen upon addition of an enzyme fraction in a second incubation would bedue to reduction of the R epimer of sulindac.

[0134] In some experiments the product was also identified by thin layerchromatography (TLC). After incubation, both the unreacted S and SSproduct were extracted into 1 ml ethyl acetate. The ethyl acetate phasewas removed, dried in a speed vacuum at room temperature and the residuewas suspended in 5 μl of ethyl acetate which was then loaded onto a TLCplate. The plate was developed with butanol:acetic acid:water (60:15:25)as the solvent. The compounds were visualized by their yellow color. TheRf values of S and SS were 0.80 and 0.95, respectively.

[0135] Results.

[0136] Using the extraction assay described above in Methods, it wasfound that recombinant MsrA from E. coli could reduce S to SS. FIG. 3Ashows a time course for the reaction and FIG. 3B shows the effect ofMsrA concentration on reduction of S. The reaction was dependent on thethioredoxin reducing system. The product, SS, was independentlyidentified by TLC.

[0137] S is a substrate for mem-R, S-Msr. E. coli is known to have atleast 6 members of the Msr family. Referring to Table 2, these proteinsdiffer in their stereo-specificity, substrate specificity, i.e., freevs. protein-bound Met(O), and location within the cell, i.e., soluble ormembrane-associated. Whereas the msrA and msrB genes have been clonedand the recombinant proteins purified, the other soluble E. coli Msrenzymes (i.e., fSMsr, fRMsr and MsrA1) have been only partiallypurified, but have been separated by conventional fractionationprocedures using DEAE cellulose chromatography (Etienne, F. et al.,Biochem. & Biophys. Res. Comm. 300 (2003) 378-382; Spector, D. et al.,Biochem. & Biophys. Res. Comm. 302 (2003) 284-289). The membraneassociated Msr (i.e., mem-R, S Msr), which has activity toward both theR and S forms of free and peptide bound met(o), was present as amembrane vesicle preparation. TABLE 2 Substrate Specificity ofMethionine Sulfoxide Reductases in E. coli ENZYME SUBSTRATE TYPEFree-R-(O) Free-S-(O) Peptide-R-(O) Peptide-S-(O) MsrA + + MsrB (+) +fRMsr + fSMsr + MsrA1 + Membrane + + + + Msr

[0138] Referring to Table 3, S was compared as a substrate for highlypurified MsrA and MsrB from E. coli and the partially purified enzymepreparations. The results showed that MsrA and the mem-R,S-Msr are ableto reduce S to SS. Very weak activity was observed with MsrAl. S was nota substrate for MsrB, which recognizes peptide-bound Met-S—(O). TABLE 3Activity of E. coli Msr Enzymes Using Sulindac as a Substrate. ENZYMETYPE UNITS OF ACTIVITY MsrA 11.3 MsrB 0 fRMsr 0 fSMsr 0 MsrA1 <0.9Membrane 5.1

[0139] Referring now to Table 4, it is seen that the membrane bound Msrof E. coli, which likely contains more than one Msr activity, reducesprimarily the R form of sulindac. In these experiments either S, whichis a mixture of the R and S epimers, or the R epimer of sulindac (seeMethods) were used as substrates. Both exhibited similar activities.Although these results support the R form being reduced, definitiveproof may require the chemical synthesis. and assay of each epimer of S.TABLE 4 Membrane Msr of E. coli Reduces Primarily the R Epimer ofSulindac. SUBSTRATE NMOLES FORMED Sulindac (R, S) 2.75 Sulindac (R) 2.41

[0140] Reduction of sulindac in mammalian (bovine) tissues. Resultsshown in Table 5 reveal that crude homogenates (S-10 fractions, seeMethods) of calf liver, kidney and brain are able to reduce S. Of thetissues tested, kidney has the highest specific activity, and brain thelowest. TABLE 5 Sulindac Reductase Activity in Calf Tissues. TISSUESPECIFIC ACTIVITY Liver 4.39 Kidney 6.53 Brain 2.31

[0141] The liver extracts were fractionated and mitochondria, S-100 andS-100 pellet (microsomes) were prepared as described in Methods. Asshown in Table 6, all three cellular fractions were able to reduce S toSS. The identity of the enzyme(s) responsible for the activity was notdetermined, but preliminary evidence indicated that MsrA was largelyresponsible, based on the observation that the addition of excessamounts of Met-S—(O) inhibited the activity in all three fractions,whereas the addition of Met-R—(O) had only a slight effect. Thus theresponsible enzyme had Met-S—(O) activity. Because free Met(O) Msrenzymes (i.e., FSMsr and FRMsr) cannot reduce sulindac (Table 2), MsrAis most likely the enzyme responsible for this activity. TABLE 6Subcellular Distribution Sulindac Reductase Activity in Bovine Liver.LIVER FRACTIONS SPECIFIC ACTIVITY S-10 4.39 S-100 6.20 Mitochondria 2.44Microsomes 1.31

Example 3 Synthesis of Sulindac Methionine Catalytic Antioxidants

[0142] As shown above, sulindac is a substrate for MsrA but not forMsrB. Sulindac contains a methyl sulfoxide moiety which is recognized byMsrA enzymes, but does not contain a N-methionine sulfoxide moiety (seeFIG. 2), the substrate recognized by both MsrA and MsrB enzymes (Table2). This example describes schemes for the chemical synthesis ofderivatives of sulindac that are improved as substrates for multiple Msrenzymes including MsrB, by modification to contain an N-substitutedmethionine, in which the methionine amino group is in peptide or amidelinkage.

[0143] Referring to FIG. 4A, compound 2a(1(Z)-5-fluoro-2-methyl-1-[4-(methylsulfinyl)phenyl]methylene]-1H-indene-3-[1-methylthiomethylenyl-2-aminoacetyl]propanoicacid) is shown. Compound 2a contains a methionine group linked throughthe amino group to the acetyl moiety of sulindac. This compound wassynthesized starting from sulindac and methionine sulfoxide methylesters as follows. To a 50 ml round bottom flask under an argonatmosphere fitted with a teflon stir bar and rubber stopper, 1.4 mmol ofsulindac was dissolved in 20 ml DMF followed by the addition of 1.5 molof methionine sulfoxide methyl ester. Dicyclohexylcarbodiimide (1.2mmol), triethylamine (2.0 mmol) and 4-dimethylaminopyridine (0.05 mmol)were placed in the reaction flask. After 12 hours, TLC analysis (75%ethyl acetate in hexanes) showed the formation of the product atR_(f)=0.29. The reaction mixture was then placed onto a 2.5 cm diameterflash column filled with approximately 6 inches of silica gel and toppedoff with a quartz sand plug. The following elution sequence was used: 5%EtOAc/Hex (250 mL), 30% EtOAc/Hex (500 mL), 50% EtOAc/Hex (250 mL) and afinal elution of 85% EtOAc/Hex (250 mL). HPLC analysis of the compound(gradient elution from 5% to 95% MeCN/H₂O over 45 min) gave a peak at22.5 min with 98% purity. Proton NMR analysis of compound 2a is shown inFIG. 8.

[0144] Another methionine derivative of sulindac, i.e., compound 3a, isgiven in FIG. 4B. A suitable scheme for the synthesis of compound 3a,with control of the α-carbon stereochemistry, is shown. In thisparticular synthetic method, the synthesis begins with commerciallyavailable sulindac (racemic form). The sulindac is converted to itsmethyl ester by treatment with diazomethane (CH₂N₂). The methyl ester isthen treated with a strong base to form the enolate, followed byquenching with N-bromosuccinimide (NBS) leading to the α-bromoester(Kita et al., J. Am. Chem. Soc. 123:3214, 2001). The ester group of thisintermediate is then selectively reduced to the primary alcohol usingdiisobutylaluminum hydride (DIBAIL-H), according to the method ofFukuyama et al., J. Am. Chem. Soc. 116:3125, 1994, to give intermediatecompound 1-3a. Compound 1-3a epoxidizes to give intermediate compound2-3a. Treatment of compound 2-3a with methyl sulfide is expected to leadto the β-hydroxysulfide compound 3-3a (Conte et al., Tetrahedron Lett.30:4859, 1989). Using para-toluene sulfonylchloride (TsCl), the hydroxylgroup in compound 3-3a is converted to the corresponding tosylate(compound 4-3a). By an extension of the method of O'Donnell (O'Donnellet al., J. Am. Chem. Soc. 111:2353, 1989), the tosylate on compound 4-3areacts with a protected diphenylimino-glycine derivative under theinfluence of a cinchona alkaloid asymmetric phase-transfer catalyst.This reaction gives the corresponding α-imino ester (compound 5-3a),with control over the stereochemistry of the α-carbon. Subsequentaqueous hydrolysis of the imino and tert-butyl ester groups gives thedesired compound 3a.

[0145] Referring now to FIG. 5A, sulindac contains a methylene groupadjacent to a carboxyl that is easily converted into enolate 1-4a.Lithium diisopropylamide (LDA) is a base typically used to form thesetypes of enolates. Intermediate 1-4a should react with bromoacetylmethionine sulfoxide (A) to form the new carbon-carbon bond found in2-4a. Hydrolysis of this intermediate with lithium hydroxide gives thecorresponding carboxylic acid derivative (compound 4a).

[0146]FIG. 5B illustrates yet another embodiment of an N-methioninederivative of sulindac indicated as compound 5a. In compound 5a, thesulindac structure and the N-acetyl methionine group are tethered by adiamine chain that can be of varying length. The use of such a linkermolecule provides the ability to generate a large variety of methioninederivatives through combinatorial synthesis methods. Compound 5a may beobtained as follows (FIG. 5B). Under the action of DCC, sulindac iscoupled to tert-butoxycarbonyl (BOC) mono-protected diamine, followed byremoval of the BOC protecting group under acidic conditions usingtrifluoroacetic acid (TFA). This intermediate is coupled to N-acetylmethionine in the presence of DCC to give compound 5a. Compound 5a caneasily be obtained as the single enantiomer (or epimers of the sulfoxideposition). The addition of N-acetyl methionine moieties is preferred, asthese moieties are expected to act as a substrate for enzymes thatrecognize N-blocked methionine sulfoxide, (such as MsrA and MsrB). Damino acids may be preferred to minimize metabolism. A racemic mixtureof the sulfoxides (i.e., both R and S forms) is preferred if it isdesired to have the compound function as a substrate for most, if notall, known Msr family enzymes that recognize free or protein-bound formsof methionine sulfoxide (whether R or S epimers).

Example 4 Synthesis of Methionine Catalytic Antioxidants Derived FromSalicylic Acid and Mefenamic Acid

[0147] This example describes chemical synthetic schemes suitable forpreparing bi-functional compounds that can serve both as catalyticantioxidants and anti-inflammatory agents (COX inhibitors).

[0148] As described above, sulindac is one example of a COX inhibitor.This example describes methionine derivatives of other COX inhibitors,i.e., acetyl salicylic acid and mefenamic acid. These bifunctionalantioxidant compounds contain the amino group of methionine in the formof an amide and preferably retain the carboxyl group found in the parentcompounds that may be critical to their inhibitory action.

[0149] Referring to FIG. 6A, starting from the methyl ester of salicylicacid, the phenol hydroxy group is shown to react with the carbon bearingthe bromine in bromoacetylmethionine sulfoxide (BAMS) to form theoxygen-carbon bond of intermediate 1-6a. In the case of mefenamic acid,the reaction with BAMS is shown to occur at the amine nitrogen to giveintermediate 1-7a (FIG. 6B). The salicyclic and mefenamic methioninesulfoxide derivatives can be converted to the respective carboxylic acidproducts 6a and 7a using a mild hydrolysis reaction with lithiumhydroxide (LiOH).

Example 5 Synthesis of Methionine Catalytic Antioxidants Derived FromIbuprofen, Indomethacin and Rofecoxib/Vioxx®

[0150] Referring now to FIG. 7, ibuprofen (FIG. 7A), indomethacin (FIG.7B), and rofecoxib/Vioxx® (FIG. 7C) each contain a methylene groupadjacent to a carboxyl or a sulfonyl group that is easily converted intoenolate, shown for intermediates 1-8a and 1-10a. Lithiumdiisopropylamide (LDA) is a typical base used to form enolates.Intermediates 1-8a, 1-9a, and 1-10a are shown to react with bromoacetylmethionine sulfoxide to form the new carbon-carbon bonds inintermediates 2-8a, 2-9a, and 2-10a. Hydrolysis of these intermediateswith lithium hydroxide gives the corresponding carboxylic acidderivatives (compounds 8a, 9a, and 10a).

Example 6 Sulindac Methionine Sulfoxide is a Substrate for MsrA and MsrB

[0151] As shown above, sulindac is a substrate for MsrA but not forMsrB. Referring to FIG. 4A, unmodified sulindac contains a methylsulfoxide moiety, but does not include within its structure a methioninesulfoxide moiety, the required substrate for Msr B enzymes. Sulindacmethionine sulfoxide (SMO), an N-acetyl methionine sulfoxide derivativeof sulindac described in Example 4 above includes both a methylsulfoxide and a methionine sulfoxide (see, for instance, compound 2a inFIG. 4A). This example demonstrates that SMO can serve as a substratefor both MsrA and MsrB enzymes.

[0152] Materials and Methods.

[0153] Synthesis of SMO. Sulindac methionine sulfoxide (SMO) wassynthesized according to the synthetic pathway described in Example 3supra. Compound 2a was used for these experiments.

[0154] Reductase assay and thin layer chromatography (TLC). Reactionmixtures were prepared in duplicate for assay of the reduction ofsulindac (S) and sulindac methionine sulfoxide (SMO). Mixtures containedin a total volume of 30 μl: 100 mM Tris-Cl pH 7.4, 15 mM DTT, 100 nmolesof S or SMO, 3 μg of MsrA enzyme, or 21 μg of MsrB enzyme. Incubationwas carried out for 2 hours at 37° C., at the end of which the duplicatesamples were combined and dried in a speed-vacuum unit at roomtemperature. The residue was suspended in 50 μl of ethanol, which wasthen loaded onto a silica gel TLC plate. The plate was developed withbutanol: acetic acid:water (60:15:25) as the solvent. The compounds werevisualized by their yellow color.

[0155] Results:

[0156] As discussed above, it is known that MsrA can reduce methylsulfoxide moieties that occur as functional groups within free andpeptide-bound methionine (i.e., Met(O)), but also within othermolecules. By contrast , MsrB can only reduce Met(O),and works best withMet(O) in peptide linkage (see Table 2). Accordingly, based on the knownsubstrate specificity of MsrA and MsrB, several different products wouldbe predicted upon reaction of sulindac and SMO with MsrA and MsrB. Forexample, because the structure of sulindac (S) contains only a methylsulfoxide (as seen in FIG. 2), reduction of S by MsrA results in SS.Reduction of S by MsrB would not be expected to generate a product, dueto the absence of methionine sulfoxide in S. In contrast to unmodifiedS, SMO includes both the methyl sulfoxide group of S as well as themethyl sulfoxide included in the methionine group (see, for example,compound 2a in FIG. 4A). Accordingly, reaction of SMO with MsrA couldgenerate several possible products having one or the other, or bothmethyl sulfoxide groups reduced, i.e.: sulindac sulfide methioninesulfoxide (SSMO), sulindac methionine (SM), or sulindac sulfidemethionine (SSM). With MsrB, however, only the methionine sulfoxideshould be reduced and the expected product is SM.

[0157]FIG. 9 shows TLC results from the various incubations, i.e.,MsrA+S (lane 1); MsrA+SMO (lane 2); MsrB+S (lane 3) and MsrB+SMO (lane4). In FIG. 9, the indicated substrates and reaction products are asfollows: S-sulindac; SS-sulindac sulfide; SM-sulindac methionine;SSM-sulindac sulfide methionine; SMO-sulindac methionine sulfoxide;SSMO-sulindac sulfide methionine sulfoxide. The positions where thesubstrates, products and standards migrate on the TLC plate areindicated by arrows.

[0158] The results of the enzyme assays demonstrate the following. Lane1 shows the presence of SS, indicating that sulindac is a substrate forMsrA. Lane 2 reveals formation of SSM, SM, and SSMO, demonstrating thatSMO is a substrate for MsrA and that both methly sulfoxide groups can bereduced. Lane 3 shows only S, demonstrating that unmodified sulindac isnot a substrate for MsrB. By contrast, lane 4 reveals that SMO is asubstrate for MsrB, shown by the formation of SM (FIG. 9) Thus it isshown that a methionine derivative of sulindac, i.e., SMO, can act as asubstrate for both MsrA and MsrB enzymes.

Example 7 Sulindac Increases Resistance to Oxidative Stress inDrosophila

[0159] This example demonstrates that sulindac, an antioxidantcontaining a methyl sulfoxide moiety, can extend the lifespan of fliessubjected to an agent known to kill flies via production of ROS.

[0160] Materials and Methods.

[0161] Paraquat is a cytotoxic compound known to form superoxideradicals intracellularly. Three different concentrations of paraquat(i.e., 2.5 mM, 5 mM and 10 mM) were tested. Flies (Drosophila) wereraised for 3 days on apple juice medium (33% apple juice, 1.7% sucroseand 2.7 mg/ml methyl paraben, a mold inhibitor, in 3.5% agar) containingvarious concentrations of sulindac or no supplement (Controls). After 3days at 25° C., flies were transferred to test vials for counting.

[0162] Results.

[0163] In the group treated with 2.5 mM paraquat, approximately 80% and25% of the flies in the untreated control group, respectively, werealive after 3 and 6 days of paraquat exposure. By contrast,approximately 95% and 60%, respectively, of the flies treated with 2 mMsulindac remained alive at the 3 day and 6 day time points (FIG. 10).Similar results were observed in the groups exposed to higherconcentrations of paraquat. For example, in groups exposed to 10 mMparaquat, the respective survival rates after 2 and 3 days wereapproximately 50% and 17% in the controls and 85% and 57% in thesulindac treated groups. These results demonstrate that administrationof a methyl sulfoxide-containing compound that is a substrate for MsrAcan lengthen the lifespan of paraquat-exposed flies. Earlier studiesshowed that over expressing MsrA enzyme in transgenic flies extendedtheir lifespan. The present data provide evidence that increasing theintracellular level of a substrate for the Msr system can also provide aprotective effect against damaging ROS species, leading to increasedlongevity under conditions of oxidative stress.

Example 8 Sulindac Promotes Cell Survival in Neuronal Cells Subjected toOxidative Damage by MPP+

[0164] This example demonstrates a protective effect of sulindac onPC-12 cells following insult with MPP+, a toxic compound thatselectively destroys dopaminergic neurons in vitro, and in an in vivoanimal model of Parkinson's disease.

[0165] Materials and Methods.

[0166] MPP+ neurotoxin. The neurotoxin 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) when given to both humans andprimates results in a clinical syndrome closely similar to Parkinson'sdisease. The compound is metabolized to 1-methyl-4-phenylpyridinium(MPP+) by monamine oxidase B and is subsequently selectively taken up bydopaminergic terminals and concentrated in the neuronal mitochondria inthe substantia nigra. MPP+ inhibits complex 1 of the electron transportchain and is thought to cause irreversible inactivation of the complexby generating free radicals (Hartley A., Stone J. M., Heron C, Cooper J.M., and Schapira A. H. V. J. Neurosci. 63:1987-1990, 1994) MPP+increases superoxide synthesis in vivo and in vitro. MPP+ damage isdecreased in transgenic mice overexpressing superoxide dismutase,suggesting that free radicals are involved in its neurotoxicity.

[0167] Cell culture. PC-12 cells were initially grown overnight inDulbecco's modified Eagle's medium containing high glucose (Gibco#11195-065), 5% fetal calf serum and 10% horse serum in 9 cm dishes. Thecells were then transferred to 6 cm dishes and grown in the same mediumwithout glucose but using sodium pyruvate (Gibco #11966-025) as the soleenergy source These cells were pretreated with sulindac (Sigma) atconcentrations of 0.1, 0.2, or 0.5 mM for 48 hours, the mediumcontaining the sulindac was removed and replaced with fresh medium Thecells were then incubated for 24 hours in medium containing MPP+ at afinal concentrations of 0.2 mM. Control cells were incubated inMPP+-free medium. At the end of the 24 hr period, cell viability wasassayed by trypan blue exclusion.

[0168] Results.

[0169] Referring to Table 7, the results show that 0.2 mM MPP+ washighly toxic to PC-12 cells, causing approximately 85% of the cells todie (15% cell survival) following a 24 hour treatment with thiscompound. Pretreatment with sulindac prior to MPP+ insult was protectiveagainst cell death, exhibiting a dose-response with approximately 35%cell death (65% cell survival) following pretreatment with the maximumconcentration tested, i.e., 0.5 mM. In the absence of MPP+, sulindac hadno effect on the viability of the cells. TABLE 7 Effect of Sulindac onthe Viability of PC-12 Cells Treated with MPP+. Sulindac (mM) Dead cells(%) Exp 1 Dead cells (%) Exp 2 0 85 87 0.1 67 74 0.2 55 39 0.5 34 35

Example 9 Sulindac Extends the Lifespan of a Transgenic Mouse Model ofFamilial Amyotrophic Lateral Sclerosis (ALS)

[0170] This example provides evidence that sulindac, a methyl sulfoxidecontaining compound that acts as a substrate for MsrA enzymes cansignificantly extend lifespan, increase motor neuron cell count andimprove motor performance in a mouse model of ALS based on a mutation insuperoxide dismutase (SOD1).

[0171] Materials and Methods:

[0172] ALS is an adult onset neurodegenerative disease of generallyunknown etiology. ALS is most commonly sporadic, with about 10% of casesbeing inherited as an autosomal dominant familial form. It is now knownthat about 20% of the familial cases are associated with a mutant formof Cu/Zn SOD (Rosen,D. R., et.al., (1993) Nature 362:59-62 ). Althoughthe protein harbors a mutation (over 100 different SOD mutations havingbeen documented in ALS patients), it is still enzymatically active.Oxidative damage is one of the main hypotheses for the toxicity of themutant protein. The animals used in this study express a mutant form ofSOD that models a mutation described in patients with ALS.

[0173] Transgenic mice expressing a mutant form of SOD similar to thatfound in human ALS patients were used for this study. Transgenic malemice with a G93A human SOD1 (G1H/+) mutation (B6SJL-TgN (SOD1-G93A)1Gur; Jackson Laboratories, Maine) were used to breed with female B6SJLmice (Jackson Laboratories, Maine). The F1 generations were genotypedfor the G93A mutation with polymerase chain reaction (PCR) using tailDNA, and two specific primers from the SOD1 gene.

[0174] Sulindac administration. G93A mice were treated with sulindac attwo different doses, i.e., 300 PPM and 450 PPM, which was mixed intotheir food beginning on postnatal day 30. Three groups were examined(i.e., 300 PPM, 450 PPM sulindac and controls). Motor performance wasassessed by Rotarod testing for each group and survival time wasrecorded.

[0175] Motor function testing. Mice were trained for 2-3 days to becomeacquainted with the Rotarod apparatus (Columbus instruments, Columbus,Ohio). Rotarod performances were assessed in G93A mice starting at 60days of age. The testing began with placing the mice on a rod thatrotates at 12 rpms. The time period that the mice stayed on the rodbefore falling off was recorded as a measurement of the competence oftheir motor function. Three trials were performed, and the best resultof the three trials was recorded representing the status of the motorperformance. Mice were tested twice a week until they could no longerperform the task.

[0176] Survival times. The initial sign of disease in G93A transgenicmice is a resting tremor that progresses to gait impairment,asymmetrical or symmetrical paralysis of the hind limbs, and ultimatelycomplete paralysis at the end stage. Mice were sacrificed when they wereunable to roll over within 20 seconds after being pushed on their side.This time point was determined to be the time of survival, at which timethe mice were sacrificed.

[0177] Light microscopic immunocytochemistry. Mice were perfusedtranscardially with cold 0.1M phosphate-buffered saline (PBS) for 1minute followed by cold 4% paraformaldehyde in PBS for 10 minutes. Thespinal cords were removed rapidly, blocked coronally, and post-fixed in4% paraformaldehyde in PBS for 6 hours. Blocks were cryo-protected in30% sucrose for 24 hours and were sectioned on a cryostat at a thicknessof 35 micrometers. All protocols were conducted within NIH guidelinesfor animal research and were approved by the Institutional Animal Careand Use Committee (IACUC).

[0178] Serial transverse sections (50 μm thick) were cut on a cryostatand collected for Niss1 staining. Every fourth section was analyzed forneuronal volume and number using the optical fractionator and nucleatorprobes of the Stereo Investigator System (Microbrightfield, Colchester,Vt.). Six tissue sections of the lumbar spinal cord from each mouse wereanalyzed. All cells were counted from within the ventral horn below ahorizontal line across the gray matter through the ventral border of thecentral canal. Photomicrographs were taken on a Zeiss Axiophot IImicroscope.

[0179] Statistical analysis. Statistical analysis of survival wasperformed using Kaplan-Meier test for survival measured in postnataldays, Fisher's Test for mean age of death analysis, and Scheffe test formotor performance.

[0180] Results:

[0181] Referring to FIG. 11, G93A mice treated with 450 PPM sulindacsurvived an average of 131.17±10.9 days. This was a 7% increase overcontrol mice, which survived an average of 123.16±11 days (P=0.083).G93A mice treated with 300 PPM sulindac also exhibited extended survival(a 10% increase) relative to the controls, with mean survival time of135.17±11.4 days (P=0.02).

[0182] The results of several statistical tests of the data shown inFIG. 11 are presented in Table 8. TABLE 8 Rank Test Chi-Square DFP-Value Logrank (Mantel-Cox) 6.744 2 .0343 Breslow-Gehan-Wilcoxon 7.7962 .0203 Tarone-Ware 7.374 2 .0250 Peto-Peto-Wilcoxon 7.661 2 .0217Harrington-Fleming (rho = .5) 7.374 2 .0250

[0183] The sulindac-treated groups showed a significant improvement inmotor performance, as evaluated by Rotarod performance times (FIG. 12).Microscopic analysis of spinal cord sections revealed that thesulindac-treated mice had significantly higher counts of motor neuronsas compared with G93A controls (FIG. 13). Differences between the 300PPM and 450 PPM sulindac groups were not significant (FIG. 13 and Table9). TABLE 9 Scheffe for Motor Performance Mean Diff. Crit Diff. P-ValueControl, Sulindac 300 PPM −59.443 54.695 .0308 S Control, Sulindac 450PPM −73.119 53.271 .0053 S Sulindac 300 PPM, −13.676 56.815 .8267Sulindac 450 PPM

Other Embodiments

[0184] This description has been by way of example of how thecompositions and methods of the invention can be made and carried out.Various details may be modified in arriving at the other detailedembodiments, and many of these embodiments will come within the scope ofthe invention. Therefore, to apprise the public of the scope of theinvention and the embodiments covered by the invention, the followingclaims are made.

What is claimed is:
 1. A non-naturally occurring compound comprising atleast one methyl sulfide or methyl sulfoxide moiety, the compound beinga substrate for at least one MsrA enzyme and at least one MsrB enzyme,or a pharmaceutically acceptable salt thereof.
 2. The compound of claim1, having formula 2, or a pharmaceutically acceptable salt thereof:

wherein: R₁ is CH of either R or S configuration; R₂ is a normal orbranched alkyl or fluoroalkyl group having 1 to 6 carbons; R₃ is methylor ethyl or a fluorinated derivative thereof; R₄ is a hydrogen or anormal or branched alkyl group having 1 to 6 carbons; R₅ is a CH ofeither R or S configuration; R₆ is a hydrogen or a normal or branchedalkyl or fluoroalkyl group having 1 to 6 carbons; R₇ is a nitrogen withsubstituent R₄ as defined herein, a CH of either R or S configuration,or a normal or branched alkyl or fluoroalkyl group having 1 to 6carbons; and X is either S or Se in any oxidation state.
 3. The compoundof claim 1, having formula 3, or a pharmaceutically acceptable saltthereof:

wherein: R₁ is CH of either R or S configuration; R₂ is a normal orbranched alkyl or fluoroalkyl group having 1 to 6 carbons; R₃ is methylor ethyl or a fluorinated derivative thereof; R₄ is a hydrogen or anormal or branched alkyl group having 1 to 6 carbons; R₅ is a CH ofeither R or S configuration; R₆ is a hydrogen or a normal or branchedalkyl or fluoroalkyl group having 1 to 6 carbons; and X is either S orSe in any oxidation state.
 4. The compound of claim 1, having formula 4,or a pharmaceutically acceptable salt thereof:

wherein: R₁ is CH of either R or S configuration; R₂ is a normal orbranched alkyl or fluoroalkyl group having 1 to 6 carbons; R₃ is methylor ethyl or a fluorinated derivative thereof; R₄ is a hydrogen or anormal or branched alkyl group having 1 to 6 carbons; R₅ is a CH ofeither R or S configuration; R₆ is a hydrogen or a normal or branchedalkyl or fluoroalkyl group having 1 to 6 carbons; and X is either S orSe in any oxidation state.
 5. The compound of claim 1, having formula 5,or a pharmaceutically acceptable salt thereof:

wherein: R₁ is CH of either R or S configuration; R₂ is a normal orbranched alkyl or fluoroalkyl group having 1 to 6 carbons; R₃ is methylor ethyl or a fluorinated derivative thereof; R₄ is a hydrogen or anormal or branched alkyl group having 1 to 6 carbons; and X is either Sor Se in any oxidation state.
 6. The compound of claim 1, having formula2a, or a pharmaceutically acceptable salt thereof:


7. The compound of claim 1, having formula 3a, or a pharmaceuticallyacceptable salt thereof.


8. The compound of claim 1, having formula 4a, or a pharmaceuticallyacceptable salt thereof.


9. The compound of claim 1, having formula 5a, or a pharmaceuticallyacceptable salt thereof.


10. A non-naturally occurring compound comprising at least one methylsulfide or methyl sulfoxide moiety, the compound being a substrate forat least one Msr enzyme, said compound having a backbone not based onsulindac(1(Z)-5-fluoro-2-methyl-1[[4-(methylsulfinyl)phenyl)methylene]-1H-indenyl-3-aceticacid).
 11. The compound of claim 10 having formula 6, or apharmaceutically acceptable salt thereof:

wherein: the aromatic ring includes one or more nitrogen atoms; thearomatic carboxyl group is oriented ortho, meta, orpara to themethionine-based moiety; R₁ is CH of either R or S configuration; R₂ isa normal or branched alkyl or fluoroalkyl group having 1 to 6 carbons;R₃ is methyl or ethyl or a fluorinated derivative thereof; R₄ is ahydrogen or a normal or branched alkyl group having 1 to 6 carbons; R₅is a nitrogen with substituent R₄ as defined herein, an oxygen, or asulfur; and X is S or Se in any oxidation state.
 12. The compound ofclaim 10 having formula 6a, or a pharmaceutically acceptable saltthereof:


13. The compound of claim 10 having formula 7, or a pharmaceuticallyacceptable salt thereof:

wherein: both aromatic rings comprises one or more nitrogen atoms; thearomatic carboxyl group is oriented ortho, meta, orpara to the anilinenitrogen; R₁ is CH of either R or S configuration; R₂ is normal orbranched alkyl or fluoroalkyl group having 1 to 6 carbons; R₃ is methylor ethyl or a fluorinated derivative thereof; R₄ is a hydrogen or anormal or branched alkyl group having 1 to 6 carbons; and X is S or Sein any oxidation state.
 14. The compound of claim 10 having formula 7a,or a pharmaceutically acceptable salt thereof:


15. The compound of claim 10 having formula 8, or a pharmaceuticallyacceptable salt thereof:

wherein: the aromatic ring comprises one or more nitrogen atoms; thesec-butyl group is oriented ortho, meta, orpara to the methionine-basedmoiety; R₁ is CH of either R or S configuration; R₂ is a normal orbranched alkyl or fluoroalkyl group having 1 to 6 carbons; R₃ is methylor ethyl or a fluorinated derivative thereof; R₄ is a hydrogen or anormal or branched alkyl group having 1 to 6 carbons; R₅ is a CH ofeither R or S configuration; X is either S or Se in any oxidation state.16. The compound of claim 10 having formula 8a, or a pharmaceuticallyacceptable salt thereof.


17. The compound of claim 10 having formula 9, or a pharmaceuticallyacceptable salt thereof:

wherein: Groups R₁, R₂, R₃, R₄, R₅, R₆, R₇ and X in general structure 9are defined as follows: R₁ is CH of either R or S configuration; R₂ is anormal or branched alkyl or fluoroalkyl group having 1 to 6 carbons; R₃is methyl or ethyl or a fluorinated derivative thereof; R₄ is a hydrogenor a normal or branched alkyl group having 1 to 6 carbons; R₅ is a CH ofeither R or S configuration; R₆ is a hydrogen or a normal or branchedalkyl or fluoroalkyl group having 1 to 6 carbons; R₇ is any halogenoriented ortho, meta, orpara to the carbonyl group, and X is S or Se inany oxidation state.
 18. The compound of claim 10 having formula 9a, ora pharmaceutically acceptable salt thereof:


19. The compound of claim 10 having formula 10, or a pharmaceuticallyacceptable salt thereof:

wherein: the lactone ring is oriented ortho, meta, orpara to thesulfonyl group; R₁ is CH of either R or S configuration; R₂ is a normalor branched alkyl or fluoroalkyl group having 1 to 6 carbons; R₃ ismethyl or ethyl or a fluorinated derivative thereof; R₄ is a hydrogen ora normal or branched alkyl group having 1 to 6 carbons; X is S or Se inany oxidation state; Ar is a phenyl, alkyl, halogen substituted phenyl,or heteroaromatic compound.
 20. The compound of claim 10 having formula10a, or a pharmaceutically acceptable salt thereof:


21. A composition comprising the compound of claim 1 and apharmaceutically acceptable carrier.
 22. A composition comprising thecompound of claim 10 and a pharmaceutically acceptable carrier.
 23. Amethod for reducing, preventing or reversing oxidative damage in a cell,the method comprising the steps of: (a) providing a non-naturallyoccurring compound comprising at least one methyl sulfide or methylsulfoxide moiety, the compound being a substrate for at least one Msrenzyme; (b) providing a cell expressing at least one Msr enzyme, saidcell comprising or being exposed to reactive oxygen species; and (c)contacting the cell with an amount of the compound sufficient to reduce,prevent, or reverse oxidative damage in the cell by said reactive oxygenspecies.
 24. The method of claim 23, wherein the cell is within ananimal subject.
 25. The method of claim 23, wherein the animal subjecthas a condition or disorder associated with oxidative damage.
 26. Themethod of claim 23, wherein the disorder involves degeneration of anerve cell.
 27. The method of claim 23, wherein the condition isage-related.
 28. A method for extending the lifespan of an animalcomprising administering to the animal a therapeutically effectiveamount of a non-naturally occurring compound comprising at least onemethyl sulfide or methyl sulfoxide moiety, the compound being asubstrate for at least one Msr enzyme.